Apparatus and technique for preservation of isolated organs through perfusion

ABSTRACT

1. APPARATUS FOR PRESERVATION OF ISOLATED VIABLE ORGANS BY HYPOTHERMIC PERFUSION, COMPRISING AN ORGAN HOLDING CHAMBER, PUMPING MEANS FOR CIRCULATING OF A PERFUSATE HAVING A FIRST CONDUIT FOR ADMINISTERING PERFUSATE TO AN ARTERY OF AN ORGAN CONTAINED IN THE CHAMBER AND A SECOND CONDUIT IN COMMUNICATION WITH SAID FIRST CONDUIT AND WITH THE INTERIOR OF SAID CHAMBER FOR SUPPLYING PERFUSATE OVER THE OUTER SURFACE OF SAID ORGAN, VALVE MEANS FOR REGULATING THE PRESSURE OF SAID PERFUSATE BEING ADMINISTERED TO SAID ORGAN, HEAT EXCHANGER MEANS FOR COOLING THE PERFUSATE, A GAS-LIQUID FILM CONTACT OXYGENATOR CONTAINED IN SAID CHAMBER, SAID OXYGENATOR COMPRISING AT LEAST ONE INCLINED FLAT HYDROPHILIC SURFACE POSITIONED TO RECEIVE EFFLUENT FROM SAID ORGAN AND PERFUSATE SUPPLIED BY SAID SECOND CONDUIT, AND MEANS FOR SUPPLYING OXYGEN CONTAINING GAS TO THE INTERIOR OF SAID CHAMBER FOR CONTACT WITH PERFUSATE ON SAID HYDROPHILIC SURFACE.

Oct. 22, 1974 M. BIER 3,843,455

APPARATUS AND TECHNIQUE FOR PRESERVATION OF ISOLATED ORGANS THROUGH PERFUSION Filed Sept. 15, 1972 s Sheets-Sheet 1 Oct. 22, 1974 Bl 3,843,455

APPARATUS AND TECHNIQUE FOR PRESERVATION 0F ISOLATED ORGANS THROUGH PIRFUSION Filed Sept. 13, 1972 3 Sheets-Sheet 2 Oct. 22, 1974 M. BIER 3,843,455

APPARATUS AND TECHNIQUE FOR PRESERVATION OF ISOLATED ORGANS THROUGH PERFUSION Filed Sept. 13, L972 3 Sheets-Sheet 5 United States Patent 3,843,455 APPARATUS AND TECHNIQUE FOR PRESER- VATION OF ISOLATED ORGANS THROUGH PERFUSION Milan Bier, 5341 E. 7th St., Tucson, Ariz. 85711 Filed Sept. 13, 1972, Ser. No. 288,499

Int. Cl. C12k 9/00 US. Cl. 195127 9 Claims ABSTRACT OF THE DISCLOSURE This invention relates to apparatus and technique for preservation of viable organs, mainly kidneys, for purposes of transplantation. The organ is perfused with a plasma or plasma derivative containing fluid at low temperature, with the organ being contained in a chamber which functions at the same time as the oxygenator for the oxygenation of the perfusate. The oxygenation is carried out on a flowing film of perfusate with direct air-liquid contact. The organ-holding chamber contains an inclined flat surface with hydrophilic properties or covered by a hydrophilic liner, which facilitates the uniform spreading of the flowing perfusate into a thin film, a current of air being directed over it. Addition of non-volatile buffers to the perfusate stabilizes the pH and avoids the necessity of providing carbon dioxide in the oxygenating atmosphere.

The invention relates to the development of a perfusion apparatus and method for preservation of viable organs for purposes of their transplantation.

Transplantation of organs is an increasingly important life-saving surgical technique. This technique is most successful with kidneys, but many other organs, including liver, heart, and lungs have been transplanted in man and in experimental animals. It is often impossible to immediately reimplant an excised organ. This is particularly true if the organ donor is not living, i.e. if the organ is removed from a recently deceased person. It is for this reason that techniques and instruments were developed for the preservation of viable organs. Another reason for the need of preservation techniques is that often the donor of the organ may be located in a different hospital than the intended recipient. With unrelated cadaver donors it is common practice to match immunological tissue compatibility between donor and recipient, and the selection of best possible match may require not only transportation between different hospitals in the same area, but also transportation from city to city. There may be also some advantages in perfusing an organ from a living donor, as a means for more thorough washing out of lymphocytes from the organ to be transplated, to minimize the immunological rejection reaction. There is a great demand, therefore, for instrumentation for organ preservation which is easily transportable by car or air.

The objective of organ perfusion is the preservation of organs in a viable state for subsequent transplantation. The only acceptable proof of successful preservation is the proper functioning of the organ upon reimplantation. As this surgical technique is usually carried out only when life itself depends on the organ function, the ultimate test is the survival of the organ recipient.

The most successful technique for the preservation of transplant organs is the hypothermic perfusion of organs with a plasma-containing solution, called perfusate, carefully adjusted to physiological requirements of the organ. The organ perfusion instruments used until now were complex and expensive. They comprised an organ-holding chamber, pulsatile pump for circulation of the perfusate, means of maintaining hypothermia, and several controls. Oxygenation was carried out by means of membrane oxy- 'ice genators, using an atmosphere enriched in oxygen and carbon dioxide, supplied from compressed gas clylinders.

A principal object of the invention is the simplification of instruments and techniques for organ perfusion, achieved by the development of a more effective oxygenator, with direct gas-liquid contact, which can utilize air only, thereby avoiding the need for membrane oxygenators and compressed gas cylinders.

Another object of the invention is to provide an organholding chamber which functions at the same time as oxygenator, air being directly blown into the organ-holding chamber.

A further object of the invention is an improved oxygenator with direct gas-liquid contact, comprising one or more inclined flat surfaces of hydrophilic nature, or covered with a hydrophilic liner, causing the fluid through said oxygenator to form a flowing film of liquid evenly distributed over the flat surface.

Another object of the invention is to incorporate into the organ-holding chamber a direct gas-liquid contact oxygenator in the form of one or more inclined fiat surfaces over which the perfusate will flow by gravity alone, said fiat surface being of a hydrophilic nature or being covered by a hydrophilic liner to permit uniform spreading of the flowing perfusate in a thin layer or film to maximize oxygenation.

Another object of the invention is an improved technique of perfusion, comprising the inclusion into the perfusate of an increased concentration of nonvolatile buffers, such as phosphate buffer, which, while compatible with physiological requirements of the organ, will help in maintaining the pH of the perfusate within the narrow limits tolerated by the organ, thereby eliminating the need of a supply of CO another characteristic feature of present instruments.

Another object of the invention is to provide continuous means of registering flow through the organ, and pressure of the perfusate going to the organ, these being important parameters for assessing viability.

Another object of the invention is to provide a readily portable apparatus, all components of which coming into contact with either the organ or the perfusate can be prestirilized and are of sufficiently low cost to be readily disposable.

Another object of the invention is to provide a system whereby the pressure of the perfusate to be delivered to the organ is maintained below a fixed maximum pressure, irrespective of changes in the resistance to flow of the organ. A further object is to provide a flow path to the perfusate such that at least part of the perfusate is continuously moistening the outer surface of the perfused organ.

Other objects and advantages of the invention will be apparent from the consideration of the specifications and claims.

The best known apparatus and technique of isolated organ perfusion, still in Widespread use, was described by Belzer et al. (Annals of Surgery 168, 38239l, 1968). It utilizes the principle of pulsatile hypothermic perfusion, a membrane oxygenator being included in the pathway of the perfusate flow for its oxygenation, and a mixture of oxygen and CO being used on the outside of the membrane oxygenator. Essentially similar devices have been constructed by a number of other investigators, for instance by Moberg et al. (Waters Instruments, Inc., Rochester, Minn.). While these instruments were effective, they were complex and costly. Some of their shortcomings and comparative advantages of the present invention can be summarized as follows:

1. Membrane oxygenators have been originally developed to prevent direct contact between air and blood, the direct air-blood interface causing damage to formed blood elements. In effect, the membrane slows down gaseous interchange, and therefore the previous perfusion instruments needed an oxygen-enriched environment on the outside of the membrane oxygenators to provide adequate oxygenation. In the present invention it has been discovered that adequate oxygenation can be obtained by air only if membranes are eliminated. Damage to perfusate does not occur as it does not contain formed blood elements. The elimination of the need for pure oxygen, usual- 1y contained in pressurized cylinders, greatly reduces the weight of the apparatus and increases its portability.

2. In all previously available instruments the oxygenator is a separate piece of equipment, adding to the cost and complexity of the apparatus. In the present invention the oxygenation is incorporated in the organ-holding chamber, thereby eliminating the need of a separate oxygenation compartment. This is achieved by providing the organholding chamber with an inclined flat surface over which the perfusate flows by gravity only, said surface being hydrophilic or covered with a hydrophiiic liner, which assures the spreading of the flowing perfusate in a thin layer to optimize gas exchange.

3. The prior technique for organ perfusion utilized the normally occurring buffer system in plasma for pH control, close pH control being essential for survival of the perfused organ. The normal buffer in plasma is the bicarbonate-carbonic acid system. The carbonic acid being volatile, it had to be continuously replaced through the membrane oxygenator. This was not only difficult, i.e. needed frequent pH monitoring to assess adequacy of carbonic acid replacement, but also required another gas cylinder of compressed carbon dioxide, adding to the weight of the apparatus. It has been discovered in the present invention that replacement of carbonic acid is not necessary and that improved pH control can be obtained by adding to the perfusate a non-volatile buffering agent. This could be a 0.001 to 0.05 molar concentration of sodium-potassium phosphate, in the pH range 7.3 to 7.5, and similar buffers are available. The pH control is rendered even more effective by first degasing the perfusate, thereby removing part of its bicarbonate, or using not plasma but plasma derivatives in the perfusate, such as purified serum albumin, which contain no bicarbonate.

4. In all existing instruments the total flow of perfusate was channeled through the perfused organ. If the resistance to fluid flow of the organ changed, due to its normal variations in capillary resistance, the pressure of perfusate would tend to change. This necessitated frequent monitoring or continuous recording of perfusate pressures. If pressure was too low, the organ became insufficiently perfused, i.e. its oxygen supply would become insuflicient because of deceased flow of perfusate through the organ, as well as decreased flow through the oxygenator. Too high pressures were also undesirable, causing edema of the organ. In the present invention this was overcome by directing only part of the perfusate flow through the organ, the bypassing remainder of the perfusate flowing over the organ. By regulating the pressure of the bypass, the pressure on the organ was also regulated. Because of the larger flow through the bypass, slight changes in capillary resistance of the perfused organ were without effect on overall pressures. This provided also greater overall rate of flow of perfusate through the oxygenator, improving its efficiency and providing a constant rate of oxygenation, irrespective of changes in perfusate flow through the organ.

5. With all perfusate flowing through the organ, there was also danger of drying out the surface of the perfused organ. The flow of the bypassing perfusate can be directed over the outside of the organ, thereby maintaining its surface in moist and more physiologic condition.

6. In existing instruments numerous controls were necessary to adjust pressure or flow rate of the perfusate, temperatures, oxygen and CO flow, etc. In the newly invented apparatus only a single control is necessary, that which regulates the flow of perfusate through the organ bypass.

The use of the apparatus is therefore greatly simplified. These and other advantages of the present invention will ecome obvious in the following description. In FIG. 1 is the flow diagram of a preferred version of the apparatus suitable for the perfusion of a single organ, in this case a kidney. FIG. 2 is a perspective view of a preferred form of apparatus according to the invention, and FIGS is an exploded view of the apparatus shown in FIG. 2.

A plastic box 1 is the organ-holding chamber, held in an inclined position by means not shown in this diagram. At least part of the bottom of the box is essentially a flat surface and this surface is covered with a hydrophilic liner 2, such as a sheet of filter paper, fiberglass filter, microporous filters of cellulose acetate, dialyzing membranes of regenerated cellulose, etc. Their purpose is to cause uniform spreading of flowing perfusate. The surface is kept inclined with respect to the horizontal so as to cause gravity flow of perfusate from the organ and the bypass inlet toward the outlet. An inclination of 5-10 with respect to the horizontal is suflicient to achieve this end. A higher inclination of up to 30, or even 45, is preferred in order to assure the gravity flow of the fluid in the desired direction even if the whole apparatus is not placed on a level surface, for instance, when the apparatus is transported in a car or plane. If the hydrophilic liner has a relatively rough surface, such as filter paper, then it serves two additional purposes: (a) it provides a non-skid surface so that the organ placed in the chamber has a lesser tendency to slide around in the chamber as a result of sudden movements as might occur during car or plane transportation; (b) the rough surface also acts as a strainer for cells and cellular debris which may be released by the perfused organ into the perfusate. During the operation of the apparatus, distinct sedimentation patterns of cells and cellular debris become clearly apparent. This is highly desirable, eliminating the need for additional filters in the system as any particulate matter in perfusate may clog the capillary bed of the perfused organ, thereby impairing its function.

The organ-holding chamber is provided by a first cover 3, used mainly for easier cleaning of the chamber, and a second cover 4, used for placing the organ, in this case a kidney 5, into the chamber. These covers can be made to snap-fit onto the chamber, or can be secured to it by adhesive tape or other similar means, including hinges.

The perfusate drains by means of tubing 6 into a perfusate-holding bottle 7, which is tightly stoppered. From it the perfusate flows into the pulsatile pump 8, of which only the compressible part is shown. This is formed by a length of flexible tubing of large diameter /2" to 1") with unidirectional flow valves 9, 10 on either end of the tubing. The compressible tubing is periodically squeezed by a cam driven by a small electric motor not shown in the diagram, this part of the apparatus being well known in the art of pulsatile pump design. The fluid flow is then directed to a heat exchanger 11, normally kept on icewater slurry, also not shown in the diagram. Following the heat exchanger, the fluid path is divided into two tubing lines 12, 13, one arm 12 going through a pressure-regulating clamp 14 into the upper part of the organ-holding chamber. This is the bypass for the perfusate, this portion of liquid flow not going through the perfused organ but its outlet being so located in the organ-holding chamber that it moistens the organ. The other arm of the tubing 13 goes through an air trap 15, a perfusate flow meter 16, and into the organ-holding chamber. In use, an organ 5 is attached to the open end of this line with a cannula leading to the artery of the organ, as is customary in the art of organ perfusion. A self-sealing sampling sleeve 17 can be located on the bypass tubing 12, permitting the withdrawal of small samples of perfusate for analytical purposes. The air trap 15 is connected to a second air trap 18, which leads to an air filter 19 and a pressure gauge 20. Two air traps are used in order to ascertain that no possible air bubbles get carried into the stream of fluid leading to the perfused organ, as air embolisms are very damaging to most organs. The air filter is of sufficiently small porosity to be sterilizing, therefore preventing the contamination of the sterile fluid pathway with any bacteria which may be contained in the pressure gauge which may not be sterile.

Above components are all the parts of the instrument coming into contact with either the perfusate or the organ and they can be manufactured cheaply enough to be disposable. In practice, the organ-holding chamber is sterilized separately. The liquid flow meter, heat exchanger, and air traps can be rigidly interconnected and can be made into an integral part to which all tubing is attached. These components are also sterilized separately. In order to assemble the instrument only a few connections need to be made: the tubing has to be attached to the three openings in the organ-holding chamber, and the air filter has to be attached to the pressure gauge. If the pressure gauge is sterilized with the rest of the equipment, the air filter 19 becomes unnecessary.

Above components describe the fluid pathway of the perfusate. Air supply is provided by a suitable air pump 21, and many such pumps are commercially available, being of the type used for aeration of fish tanks and other similar purposes. The output of the air pumps is channeled by means of tubing through a flow meter 22, into a sterilizing air filter 23, and into the organ-holding chamber.

While the above describes a preferred arrangement for fluid flow of the perfusion apparatus, obviously other elements can be added at will, such as temperature and pressure transducers, filters, etc., as is well known in the art of fluid monitoring and processing. Nor are all elements indispensible, such as the two air traps 15, 18, which could be substituted by a single air trap or air trap-filter combi nation, many of this type of device being readily commercially available as a result of general technology of supplying air-bubble-free liquids as, for instance, in sets used for parenthal administration of saline, glucose, etc.

It is obvious that the above drawing illustrates only one type of organ-holding chamber, incorporating the novel principles. Other types can be readily designed. With kidneys, for instance, it might be desirable to have a two-kidney-holding chamber, in which case the two kidneys could be placed either side by side, in a chamber roughly twice as wide, or on top of each other, if a suitable shelving arrangement is provided. It is also possible to modify in a variety of ways the spatial arrangement between the organ and the hydrophilic flat surface of the oxygenator. For instance, the kidney could be supported by a suitable shelving arrangement, from where the perfusate could be dripping on the hydrophilic surface underneath it. Nor is it necessary that the whole hydrophilic surface be in a single plane, as the filter paper could be pleated in a zig-zag fashion under the kidney, thereby reducing the surface occupied by the organ-holding chamber and increasing its height.

A complete apparatus for the organ perfusion, embodying above-described flow diagram, is illustrated in FIGS. 2, 3. The base of the apparatus is a thermally insulated container 24, which can hold a slurry of ice and water for the purpose of maintaining the necessary hypothermia. A drain is provided for emptying the water. This container has a ridge 26 which can hold the platform 27 on which are mounted most of the mechanical components of the apparatus. These consist of a small electric motor 28 rotating an eccentric cam 29 which periodically pushes on a bar 30, squeezing the compressible element of the pump 8, thereby causing pulsatile circulation of the perfusate through the apparatus. The air pump 21 causes air flow through the flow meter 22 into the top of the organ-holding chamber 1, through a sterilizing air filter 23. An ice chute 31 serves for addition of ice and is shown in its open and closed positions. The heat exchanger 11 is dipping in the ice-water slurry, and is connected on the one end to the pulsatile pump element 8, and on the other end to a Y-type connector 32. One leg of this Y-connector is the bypass for the perfusate. The elastic tubing is threaded through a pressure-regulating clamp 14 and goes to the upper hole in the organ-holding chamber. The other leg of the Y-connector feeds into a first air trap 15 and goes from there through the flow meter 16 and into the bottom hole of the organ holding chamber. The air trap 15 is connected to a second air trap 18 and from there it is connected to a pressure reading gauge 20. The perfusate is contained in the bottle 7. Two handles, not shown in the diagram, can be attached to the insulated container 24 to permit easy transport of the apparatus. The Whole apparatus can be covered with a suitable enclosure of rigid or flexible material. While in transit the apparatus can be powered by a self-contained rechargeable battery with an alternator to provide 115 volts, to operate the air and perfusate pumps. These are readily obtainable in commerce and not shown in the drawing. Electrical contact is established through the male contactor 33, which can be reached through a hole 34 in the insulated container. The organ-holding chamber is located on top of a sliding plate 35 which fits into appropriate grooves on the sides of the platform 36, 37

The assembly of the apparatus is very simple as it consists of only a few readily identifiable major components. The first component is the insulated container 24, into which the platform 27 can be readily placed on the supporting ridge 26. This platform contains rigidly attached to it all the permanent parts of the apparatus except for those which come into contact with either the perfusate or the organ, i.e. the components illustrated in drawing 1. The next step in the assembly is placing into the apparatus the presterilized components of FIG. 1, except for the kidney-holding chamber. The bottle 7 is filled with the perfusate and the pump primed by a few hand compressions of the compressible pump element 8. The sliding plate 35 is then positioned and the organ-holding chamber placed on top. This can be secured by means of adhesive tape or similar devices. The necessary connections of tubing outlets for perfusate flow, return and air supply complete the arrangement. Liquid has to be also drawn into the air traps, to approximately the middle of the upper air trap, thereby forming a double protective lock against air embolisms. Following placement of the organ in the chamber, the flow can be established, and the only regulation necessary is the clamping or releasing of the bypass clamp which regulates the maximum pressure to which the organ will be exposed during perfusion. Ice-Water slurry can be added as necessary.

It is obvious that above description exemplifies only one of the many possible instruments embodying the invention. For instance, the organ-holding chamber can be placed on the same level as the air pump and pulsatile pump, rather than above them, as shown, simply by extending the basic instrument platform 27 and using a larger insulated container. It is also possible to use a smaller insulated container, just around the heat exchanger, and house the whole unit in an appropriate box as is customary in instrument design. In the described arrangement, ice-water slurry has been chosen for cooling, as calculations have shown this to be the most effective way to maintain the desired hypothermia, in terms of total weight of apparatus. If desired, mechanical or electronic refrigeration could easily be added, and there are many such devices readily available in commerce.

The following general specifications as to dimensions, flow rates, and other conditions can be given. The organholding chamber should be large enough to contain the organ and provide sufficient surface for adequate oxygenation. With a single kidney, the overall size of the chamher is 12 x 6", with height of 3" in the organ-holding part of the chamber, this height being reduced to only /2" to l" in the remainder of the chamber, serving principally for oxygenation purposes. The overall flow rate of perfusate should be in excess of ml./minute, preferably in the range of 300 to 500 ml./minute. The heat exchanger should be of adequate length to provide chilled perfusatc in the temperature range of 4 to 14 C., the optimum temperature being in the range of l12 C. Air flow by the air pump should be at least 0.5 lt./min., and be sufficient to maintain partial oxygen pressure in the perfusate in the range of 120 to 160 p. 0 There is a general concensus that pulsatile flow of perfusate is to be preferred, mimicking the normal pulsatile flow of blood in vivo. The pumping arrangement described can be adjusted to provide a variety of pulse rates and pulse profiles by varying motor speed and shape of cams. The maximum pressure at the peak of the pulse seems to be also of consequence, and should be regulated to between 40 and 100 mm. mercury pressure at the point of delivery to the perfused organ. This regulation is accomplished by opening or closing the bypass clamp 14 in above diagrams.

Preparation of the perfusate is critical for the successful preservation of organs. All experiments reported here have been carried out with dog kidneys, and dog plasma has been used in most, but not all, perfusates. Up to the point of addition of the non-volatile buffers, the preparation followed that customary in the art of kidney perfusion. This comprises preparation of citrated dog plasma, its freezing and thawing. The thawed plasma is then filtered to remove cryoprecipitated proteins and lipoproteins. For each 850 ml. of plasma, 150 ml. of water is added to reduce protein content and the pH is adjusted to 7.4. The following additions are also considered important: 4 meq./lt. of MgS'O 0.35 to 0.40 gms./lt. of mannitol, and sufficient sodium chloride and potassium chloride to bring the ionic content of sodium to about 150 meq./lt., and that of potassium in the range of 4 to 8 meq./lt. Various other additives, such as insulin, penicillin, and other pharmaceuticals, have been advocated at one time or another, and they are all compatible with the present system. The total concentration of all solutes should correspond to 300-350 milliosmols.

The addition of non-volatile buffers is not essential for the success of the transplantation, its only effect being a better control of pH during perfusion. Several buffers could be employed, but the first choice made was that of sodium-potassium phosphate, pH 7.4, phosphates being known to be compatible with living organisms, and to be elfective buffering agents in the desired pH range of 7.3 to 7.5. Concentrated buffer was added to the adjusted perfusate so as to increase the phosphate by various increments, ranging from 0.001 to 0.05 molar concentration. All these were found to be effective in stabilizing the pH during perfusion. Better stabilization of pH is also obtainable if the initial plasma is degased by exposing it to a vacuum for about minutes, this removing substantial amounts of volatile bicarbonate. The final step in preparation of perfusate is its filtration through a sterilizing filter of 0.22 microns porosity.

This is by no means the only composition of perfusate which is necessarily compatible with survival of perfused organs, and above description should not be considered as limiting the apparatus to the use of this perfusate only. In fact, several other buffers could be used, such as N tris (hydroxymethyl) methyl-Z-aminoethane-sulfonic acid, with a pK of 7.5, N-(2-Acetamido)glycine, with a pK of 7.7, etc., the only requirement being their compatibility with living tissue and a disassociation constant falling in the range of pK values 6.6 to 8.2. Nor is the use of plasma mandatory, and one of the experiments will describe the use of human serum albumin as the sole protein in the perfusate, the other components being adjusted to similar levels as in the above described preparation. As the art of organ perfusion advances, it is certain that better definition of most suitable perfusate composition will result, and that it will be probably compatible with above described apparatus.

The experiments to be described have shown that oxygenation by air only is sufficient for maintenance of adequate oxygenation. If desired, however, the atmosphere in the kidney-holding chamber can be manipulated at will, by adding oxygen, carbon dioxide or any other gas in metered quantities to the output of the air pump, or substituting the air pump output with any other desired gas.

EXAMPLE I This example will show that the apparatus and technique described are adequate for 24 hour preservation of isolated dog kidneys in a viable state, and that the sopreserved kidneys upon implantation into nephrectomized dogs will maintain the dogs alive with adequate kidney function for at least 6 days post implantation. Several modifications of apparatus were used, all embodying the flow diagram and components illustrated in drawing 1. The kidneys to be perfused were obtained by unilateral nephrectomy. All perfusates contained dog plasma, adjusted as described, some of the variable factors in their composition being listed in table I. Following removal TABLE I.COMPOSITION OF PERFUSATE M 1 Beginning of perfusion End of perfusion phos- Na K LDH, Na K LDH Dog phate meq.l meqJ units, meqJ meq-l units N0. added It. it. ml. It. It. ml.

1 0. 03 153 7. 4 80 155 7. 5 460 2".-- 0.03 159 7.7 80 159 7. 9 180 3- 0. O1 167 8. 5 80 161 8. 6 418 4- 0. 01 185 8. I 78 177 9. 1 300 5 0. 007 178 8. 3 101 178 8. 7 300 6 0.0 180 5. 5 98 17E 6. 2 460 7. 0. 002 179 8. 7 108 175 8. 1 282 of the kidneys, they were rapidly chilled by flowing through them chilled, adjusted, lactated Ringer solution, as is customary in the art of kidney preservation. Upon connection to the perfusion apparatus the maximum pulsatile pressure was adjusted to fall within the range of 50 and 60 mm. Hg, and the pH of perfusate checked during the first hour, 8 hours afterwards, and on the end of perfusion. The pH was maintained within the range of pH 7.3 to 7.5, by adding small amounts of dilute sodium hydroxide or acetic acid, if necessary. Air flow was maintained at 1 lt./min. Samples of perfusate were withdrawn at beginning and end of perfusion and analyzed for sodium ion, potassium ion and the enzyme lactic acid dehydrogenase (LDH). Increases of potassium ions and of the enzyme content during perfusion are useful indices of kidney damage having occurred during perfusion to those skilled in the art. At the end of the perfusion the dogs second, remaining kidney was removed, and the perfused kidneys were reimplanted into the necks of the animals from which they were originally removed by anastomosing the kidney artery to the carotid artery of the dog, and the kidney vein to the jugular vein. Blood samples were taken from the dogs during the following TABLE IL-CREAIININE LEVELS IN DOGS FOLLOWING V TRANSPLANTATION Mg. creatinine per ml. plasma Day Day Day Day Day Day Day Dog number 0 1 1 2 3 4 5 6 1 Day of kidney reimplantation.

S or 6 days and the plasma content of creatinine' determined, this being the usual criterion of kidney function. These data are reported in Table II. There is usually a transient increase in creatinine, returning towards normal when the transplanted kidney assumes its full function. These data are reported on Table II. There is usually indication of proper kidney function, reinforced by the creatinine data.

9 EXAMPLE n The customary preparation of perfusate, outlined in the preceding pages, utilizes cryoprecipitate-free filtered plasma. Such a product is not commercially available and is cumbersome to prepare. The following example will show that it is possible to substitute plasma with a purified plasma derivative, serum albumin, and still obtain survival of {perfused kidney, with adequate kidney function. The experiment was conducted with a dog kidney, obtained as outlined in Example I, and perfused with a solution containing commercial human serum albumin as its only'major protein component. Human albumin was chosen, rather than the corresponding protein of canine origin, because of its ready commercial availability. It is'significant that survival of the kidney was obtained, evei in the face of this species difference.

The perfusate contained of human serum albumin, and its ionic composition was adjusted to 152 meq./lt. sodium 10118, 6.6 meq./lt. potassium ions, 105 meq./lt. chloride iqns, 4 meq./lt. magnesium sulfate, 20 meq./lt. phosphate ions, and 6 meq./lt. of calcium ions. Sodium chloride and sodium acetate were used to adjust simultaneously chloride and sodium ion concentrations, and 0.5% of mannitol was added to increase total osmolality of the perfusate to the 300-330 milliosmols range. The perfusate was adjusted to pH 7.4 and used in the same manner as described in Example I. The initial lactic acid dehydrogenase level was zero, purified human serum albumin containing none of this enzyme. Following 24 hours perfusion, the enzyme level rose to only 78 units/ml. As in Example I, creatinine levels in the dog, postoperatively, are taken as index of kidney function and reported in Table IH.

TABLE III Creatinine levels in transplanted dog, following kidney perfusion using human serum albumin Day OOQUI 0 EXAMPLE III A striking observation during the perfusion described in Example II was that the perfusate remained unusually constant, not varying by 0.1 pH units throughout the '24 hours perfusion. This is due to complete absence of hicarbonate in the perfusate. With plasma in perfusates, the pH is drifting slowly, necessitating several minor adjustments in the first hour following the beginning of perfusioi and one additional adjustment of about 0.1-0.2 pH units in the following 8 hours of perfusion. The drift is towards more alkaline values as a result of loss of volatile carbon dioxide. To the contrary, in a single experiment where perfusion was attempted with a mixture of oxygen and carbon dioxide in the oxygenator, the pH was found to drift rapidly within the pH range of pH values 7 to 7.6, depending whether there was excess or lack of adequate replacement of volatile carbon dioxide, the pH needing careful monitoring throughout the perfusion period.

I claim:

1. Apparatus for preservation of isolated viable organs by hypothermic perfusion, comprising an organ holding chamber, pumping means for circulating of a perfusate having a first conduit for administering perfusate to an artery of an organ contained in the chamber and a second conduit in communication with said first conduit and with the interior of said chamber for supplying perfusate over the outer surface of said organ, valve means for regulating the pressure of said perfusate being administered to said organ, heat exchanger means for cooling theperfusate, a gas-liquid film contact oxygenator contained in said chamber, said oxygenator comprising at least one inclined flat hydrophilic surface positioned to receive efiiuent from said organ and perfusate supplied by said second conduit, and means for supplying oxygen containing gas to the interior of said chamber for contact with perfusate on said hydrophilic surface.

2. Apparatus for preservation of isolated viable organs as claimed in claim 1, wherein said inclination of said flat surface of the oxygenator is within 5 and 45 with respect to the horizontal, causing gravity-driven flow of perfusate along this surface.

3. An apparatus for preservation of isolated viable organs as claimed in claim 2, wherein said hydrophilic liner covering said inclined flat surface of the oxygenator is constituted by a sheet of ultrafiltration membrane.

4;. An apparatus for preservation of isolated viable organs as claimed in claim 1, wherein said valve means is adjusted so as to maintain the maximum pressure of the flowing perfusate reaching the perfused organ in the range of 40 to mm. mercury pressure above atmospheriic pressure.

5,. An apparatus for preservation of isolated viable organs as claimed in claim 4, wherein a flow meter is positioned so as to measure the stream to perfusate being directed to the artery of the perfused organ.

6. An apparatus for preservation of isolated viable organs as claimed in claim 5, wherein the source of said oxygen containing gas is air.

7. An apparatus for preservation of isolated viable organs as claimed in claim 6, wherein air traps are positioned so as to entrap any air bubbles in the perfurate stream before they reach the perfused organ.

8. An organ-holding chamber for preservation or perfused organs, wherein said chamber contains means for supplying circulating perfusate to the artery of said perfused organ, means for supplying additional perfusate to the outer surface of said perfused organ, means for draining perfusate from the chamber, and an essentially flat surface held in inclined position with respect to the horizontal to support an organ held in said chamber, said flat surface being hydrophilic or being covered with a hydrophilic liner, said liner being a sheet of filtering material or a sheet of dialyzing, ultrafiltration or reverse osmosis membrane, said perfusate flowing over said fiat surface and its liner by virtue of gravity, and means for administering a flow of oxygen containing gas over said flat surface, establishing a direct gas-liquid contact between said gas and said perfusate.

9. An organ-holding chamber for preservation of perfused organs as claimed in claim 8, wherein said flat surface having a hydrophilic character or being covered by a hydrophilic liner is not in a single plane, but is pleated in a zigzag fashion, each essentially flat portion of said pleated surface being inclined with respect to the horizontal.

References Cited UNITED STATES PATENTS 3,632,473 1/1972 Belzer et a1. -127 3,738,914 6/1973 Thorne et a1. 195'-l27 3,070,092 12/1962 Wild et al. 23258.5 3,547,591 12/1970 Torres 23258.5 3,142,296 7/1964 Love 23258.5 3,607,646 9/1971 Roissart 195127 3,545,221 12/1970 Swenson et al. 195-127 ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 

1. APPARATUS FOR PRESERVATION OF ISOLATED VIABLE ORGANS BY HYPOTHERMIC PERFUSION, COMPRISING AN ORGAN HOLDING CHAMBER, PUMPING MEANS FOR CIRCULATING OF A PERFUSATE HAVING A FIRST CONDUIT FOR ADMINISTERING PERFUSATE TO AN ARTERY OF AN ORGAN CONTAINED IN THE CHAMBER AND A SECOND CONDUIT IN COMMUNICATION WITH SAID FIRST CONDUIT AND WITH THE INTERIOR OF SAID CHAMBER FOR SUPPLYING PERFUSATE OVER THE OUTER SURFACE OF SAID ORGAN, VALVE MEANS FOR REGULATING THE PRESSURE OF SAID PERFUSATE BEING ADMINISTERED TO SAID ORGAN, HEAT EXCHANGER MEANS FOR COOLING THE PERFUSATE, A GAS-LIQUID FILM CONTACT OXYGENATOR CONTAINED IN SAID CHAMBER, SAID OXYGENATOR COMPRISING AT LEAST ONE INCLINED FLAT HYDROPHILIC SURFACE POSITIONED TO RECEIVE EFFLUENT FROM SAID ORGAN AND PERFUSATE SUPPLIED BY SAID SECOND CONDUIT, AND MEANS FOR SUPPLYING OXYGEN CONTAINING GAS TO THE INTERIOR OF SAID CHAMBER FOR CONTACT WITH PERFUSATE ON SAID HYDROPHILIC SURFACE. 